In recent years the availability of automated DNA synthesizers has revolutionized the fields of molecular biology and biochemistry. As a result, linear DNA oligonucleotides of specific sequences are available commercially from several companies. Commonly, oligonucleotides are labeled after synthesis to facilitate detection and quantification of oligonucleotide-target complexes. Oligonucleotides (labeled or unlabeled) can be used for a variety of applications. For example, DNA oligonucleotides can be used as primers for cDNA synthesis, as primers for the polymerase chain reaction (PCR), as templates for RNA transcription, as linkers for plasmid construction, and as hybridization probes for research and diagnostics.
Synthesis of oligonucleotides using standard solid-phase synthetic methods is, however, expensive. Reasons for this include the high costs of the synthetically modified monomers, e.g., phosphoramidite monomers, and the fact that up to a tenfold excess of monomer is used at each step of the synthesis, with the excess being discarded. Costs of DNA oligonucleotides have been estimated at $2-5 per base for one micromole (about 3 mg of a 10 mer) on the wholesale level. On this basis, 1 gram of a 20-base oligomer would cost on the order of $20,000.
Enzymatic methods for oligonucleotide synthesis use DNA or RNA nucleotide triphosphates (dNTPs or NTPs) derived from natural sources as the building blocks and have the potential for lowering the cost of oligonucleotide synthesis. These monomers are readily available, and are less expensive to produce than phosphoramidite monomers because the synthesis of nucleotide triphosphates from base monophosphates requires as little as one step. See, for example, E. S. Simon et al., J. Org. Chem., 55, 1834 (1990). Nucleotide triphosphates (NTPs) can also be prepared enzymatically. The polymerase enzymes used in these methods are efficient catalysts, and are readily available.
The two primary methods currently used for enzymatic synthesis of DNA are cloning (e.g., J. Sambrook et al., Molecular Cloning; 2nd ed.; Cold Spring Harbor Press, 1989) and the polymerase chain reaction (e.g., R. K. Saiki et al., Science, 239, 487 (1988)). Cloning requires the insertion of a double-stranded version of the desired sequence into a plasmid, followed by transformation of a bacterium, growth, plasmid re-isolation, and excision of the desired DNA by restriction endonucleases. This method is not feasible for large-scale preparation of oligonucleotides because most of the material produced (the vector) is in the form of unusable DNA sequences.
Polymerase chain reaction (PCR) is a newer technique that uses a thermostable polymerase to copy duplex sequences using primers complementary to the DNA. Subsequent heating and cooling cycles accomplish the efficient amplification of the original sequence. However, for short oligomers, such as those used in anti-sense applications (e.g., less than about 50 nucleotides), PCR is inefficient and not cost-effective because it requires stoichiometric amounts of primer; i.e., a primer for every new strand being synthesized.
Recently, a method was developed for the enzymatic synthesis of DNA oligomers using a noncleavable linear hairpin-shaped template/primer in a PCR-like enzymatic synthesis. See G. T. Walker et al., PNAS, 89, 392 (1992). Although this method may be more cost-effective than PCR, the polymerase must still dissociate from the template to enable amplification. Furthermore, the end groups of the DNA produced are ragged and not well-defined.
Other methods of DNA replication are discussed in Harshey et al., Proc. Nat'l. Acad. Sci., USA, 78, 1090 (1985); and Watson, Molecular Biology of the Gene (3rd Edition). Harshey et al. discuss the theoretical method of "roll-in" replication of double-stranded, large, circular DNA. The "roll-in" process involves small, double-stranded circle cleavage and incorporation into a genome. It is primarily a process for inserting double-stranded plasmids into a double-stranded genome. Although one could conceivably use an entire genome to replicate an oligonucleotide, the resulting product would be thousands of nucleotides longer than desired. Thus, the "roll-in" process would be a very inefficient means to produce target oligonucleotide sequences. Watson briefly mentions the replication of single-stranded circles, but the author focuses on the replication of double-stranded circles.
Known methods for processive rolling-circle synthesis of DNA involve the extension of an oligonucleotide primer hybridized to a double-stranded circular polynucleotide template. Prior to the present invention, it was believed by those skilled in the art that processive rolling-circle synthesis would not proceed without additional proteins which unwind the duplex ahead of the polymerase. See, e.g. Eisenberg et al., PNAS USA, 73:3151 (1976); The Single-Stranded DNA Phages, D. T. Denhardt et al., eds., Cold Spring Harbor Press; Cold Spring Harbor (1978); and DNA Replication, W. H. Freeman, San Francisco, 1980. In Eisenberg et al., the in vitro replication of .phi.X174 DNA using purified proteins is disclosed. Among the listed necessary proteins are DNA unwinding protein (also known as SSB, single-strand binding protein), cisA protein, and rep protein. These DNA unwinding proteins (which also require ATP) are necessary for this replicative synthesis; otherwise the polymerase stalls.
The Single-Stranded DNA Phages (D. T. Denhardt et al., eds., Cold Spring Harbor Press; Cold Spring Harbor (1978)) includes a discussion of the mechanism of replication of a single-stranded phage. An early stage of replication involves the elongation of a single-stranded (-) template annealed to a full-length linear (+) strand. Further elongation reportedly requires unwinding of the helix ahead of the polymerase. DBP (double-strand binding protein) was believed to be necessary to coat the displaced strand in order for there to be successful DNA synthesis during elongation.
Enzymatic synthesis of DNA requires the use of an effective polymerase. For example, the polymerase from phage .phi.29 is known to amplify DNA strands as large as 70 kb in length and exhibits a high degree of processivity. However, the use of phage .phi.29 polymerase to amplify a selected oligonucleotide present as part of a typical plasmid vector still results in the wasteful (in both time and monetary resources) production of unwanted DNA sequences. Additionally, after amplification is complete, the investigator must separate the strands, cleave the product, and purify the oligonucleotide of interest from thousands of other unwanted base pairs.
RNA oligomers are currently synthesized by two principal methods: automated chemical synthesis and enzymatic runoff transcription. An automated synthesizer can be used to construct RNA oligomers using a modification of the phosphoramidite approach. See, for example, S. A. Scaringe et al., Nucleic Acids Res., 18, 5433 (1990). Chemical synthesis of RNAs has the advantage of allowing the incorporation of nonnatural nucleosides, but the yield decreases significantly as the length of the RNA product increases. Stepwise yields of only 97.5% per round of synthesis are typical. Moreover, because of the need for additional protecting groups, RNA phosphoramidite monomers are considerably more expensive than DNA phosphoramidite monomers, rendering RNA synthesis by this method extremely costly.
An alternative, the enzymatic runoff transcription method, utilizes a single or double-stranded DNA template for RNA synthesis. Runoff transcription requires a phage polymerase promoter, thus a DNA strand approximately 20 nucleotides longer than the desired RNA oligomer must be synthesized. There are also strong sequence preferences for the RNA 5' end (J. F. Milligan et al., Nucleic Acids Res., 15, 8783-8798 (1987)). In runoff transcription the RNA copy begins to form on the template after the phage polymerase promoter and runs until the end of the template is reached. This method has the disadvantages of producing RNA oligomers with ragged, ill-defined end groups and giving relatively slow amplification. Both chemical synthesis and runoff transcription produce a number of undesired products shorter or longer than the desired RNA, lowering effective yields and requiring careful purification.
Self-processing of nucleic acids can generate polynucleotides with well-defined ends. For example, double-stranded DNA plasmid vectors can be constructed to encode ribozymes as well as their associated self-cleavage sites, leading to self-processing after transcription (A. M Dzianott et al., Proc. Natl. Acad. Sci. USA, 86, 4823-4827 (1989); C. A. Grosshans et al., Nucleic Acids Res., 19, 3875-3880 (1991); K. Taira et al., Nucleic Acids Res., 19 5125-5130 (1991)). However, conventional plasmid vectors are highly inefficient templates for preparative, in vitro ribozyme synthesis because they generate significantly longer RNAs than desired. Their large size poses additional problems for delivery into cells in cases where transcription is to be performed intracellularly. Plasmid vectors also require promoters to initiate transcription.
Currently, multimeric oligonucleotides are typically synthesized by end-to-end ligation of members of a homogenous population of oligonucleotides produced by one of the above-described methods. This method results in the synthesis of complementary multimers as well, yielding double-stranded, but not single-stranded, multimeric products.
There exists a continuing need for less expensive, faster, and more efficient methods for the large-scale production of DNA and RNA oligomers and multimers, particularly single-stranded oligomers and multimers. For example, potential therapeutic applications require large amounts (tens or hundreds of grams) of well-characterized oligomers for animal and clinical trials, and even more for eventual use as pharmaceuticals. See, for example, I. Kitajima et al., Science, 258, 1792 (1992), and M. Z. Ratajczak et al., PNAS, 89, 11823 (1992). For medical and diagnostic applications involving the use of labeled oligonucleotides, a particularly useful method would allow for concurrent incorporation of the label during oligonucleotide synthesis.